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College Genetics Help

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SkierPT
Posts: 2176 - Karma: 4,720
I am taking college genetics 1 at my college and we have the rest of the semester to work on an individual cloning project. We have to clone and express our gene of interest in the pcDNA 3.1. I have no clue how to do this. There is obviously more information in my experiment packet but if anyone can help me shoot me a PM and I'll go into more detail. Hopefully theres a genetics/lab major on here somewhere.

Thanks
Major Karma and Feedback for helping a sinking college student.
Oct 28 2012 7:14PM
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a_pla5tic_bag
Posts: 9210 - Karma: 5,357
I'm here... so, you're actually cloning a gene right? you've got one of these http://products.invitrogen.com/ivgn/product/V79020?ICID=search-product
and i'm assuming you're going to be using bacteria to clone the gene? there's a positive selection marker for antibiotic resistance in there for E. coli. so i'd imagine you'll be using the bacteria to clone the gene then killing all the ones that don't have the stuff you need with antibiotics.

or you could do PCR?

Check your lab handout thing, i'm sure it's got detailed instructions on how to do everything.
Oct 28 2012 7:36PM
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Powder*Hound
Posts: 51 - Karma: 16
I work in a bio lab and clone genes on a daily basis. I agree with everything in this previous post. If you have the invitrogen kit the process is very easy. Do you already have a specific gene you have to do and primers that go with it?
Oct 28 2012 7:55PM
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SkierPT
Posts: 2176 - Karma: 4,720
I have the gene of interest which is gfp...aka pglow. The promoter is CMV and there is a list of the nucleotide sequence of the DNA showing where the cloning and restriction sites. I stiill have no idea where to start and what the steps are to achieve this. I mean It lists the steps sort of but I need a dumbed down version
Oct 28 2012 11:39PM
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squibble
Posts: 4569 - Karma: 104
google polymerase chain reaction aka pcr
Oct 28 2012 11:42PM
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a_pla5tic_bag
Posts: 9210 - Karma: 5,357
So where is the gene right now? In a plasmid? Did you get it in bacteria yet? Your lab manual has to have instructions in it.
Oct 29 2012 12:37AM
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icon1234
Posts: 3081 - Karma: 1,475
OP I don't get how you don't have a protocol for this experiment? Like does your assignment include making your own protocol as well? Like we don't have all the information to give advice.

Oct 29 2012 12:44AM
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Wompuscat
Posts: 818 - Karma: 380
I remember doing this lab in Bio II. Isn't this where you distinguish the bacteria that had pGLO and those that dont by treating with ampicillin and then using a blacklight? I may be completely wrong, I havent taken genetics yet, but that was one of my favorite labs.
Oct 29 2012 1:16AM
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SkierPT
Posts: 2176 - Karma: 4,720
Heres the experiment outline:

1. consult the multiple cloning sites described on pages 3-4 to design a stradegy to clone my GFP gene into pcDNA 3.1
2. Ligate your insert into the appropriate vector and transform into E.coli. Select transformants on LB plates containing 50 to 100 ug/ml ampicilin.
3. Analyze your transformants for the presence of insert by restriction digestion.
4. Select a transformant with the correct restriction pattern and use sequencing to confirm that your gene is cloned in the proper orientation
5. Transfect your construct into the mammalian cell line of interest using your own method of choice. Generate a stable cell line if desired
6. Test for expression of your recombinant gene by western blot analysis of functional assay

HELPPPPPPPPPP
Oct 29 2012 11:00AM
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Wompuscat
Posts: 818 - Karma: 380
yeah i havent done the sequencing to be positive the gene is there, nor have I done the western blot. But if this is a lab isn't it a wee bit difficult for us to help you man? I suggest going to a TA or professor. Just my advice. If i was in genetics Id help, I love it, but thats not til next semester.
Oct 29 2012 11:25AM
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a_pla5tic_bag
Posts: 9210 - Karma: 5,357
Hmm. well i'd certainly consult the sites it lists in your instructions. Do you know what you can use as a vector? virus? plasmid? You could just put the segment you want into a medium with bacteria and they might take it up that way. though they'd probably use their own restriction enzymes to destroy it. i bet if you had a lot of E. coli then some of them would take it up. then you could pure culture the ones expressing GFP and go from their? It says it wants you to ligate it in though. you have restriction enzymes that will cut at specific spots that will create an area for ligation. I think those are in your kit.
Oct 29 2012 12:13PM
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SkierPT
Posts: 2176 - Karma: 4,720
Well I have lab now I'll get back to you guys. We are using plasmids btw
Oct 29 2012 12:40PM
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icon1234
Posts: 3081 - Karma: 1,475
OP while I do have faith that we could prob figure out dilemma. In my experience you are much better off going to your TA or your professor. Way more efficient use of time, and you will know 100% what is expected and what you are doing is right or not.
Oct 29 2012 6:59PM
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a_pla5tic_bag
Posts: 9210 - Karma: 5,357
remembered this thread and it appears as though a week has passed since you had lab. You figure it out? I'd imagine if you had the plasmid made, or made it, then you just need to get it into the bacteria and let them start conjugating. I'm sure you know, but try and keep the medium they're in REALLY still. Conjugation connections are super weak and just tapping the medium will unhook two bacteria. Apparently it takes like 90min for everything to go across, the plasmid would probably go before 90min but that would ensure it would go. probably, i mean i'm sure you also know bacteria have a habit of just destroying new genetic info when it enters the cell.
Nov 5 2012 10:52AM
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SkierPT
Posts: 2176 - Karma: 4,720
Okay update time. So we ran the pcr and precipated out the plasmid....we did two RE digests to cut out the gene we wanted. Now we have to set up a large scale prep of the DNA we have. We do two more RE digests of that and then somehow insert the two together? Does this sound right?
Nov 10 2012 12:22AM
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a_pla5tic_bag
Posts: 9210 - Karma: 5,357
So you have the gene of interest in a plasmid and said plasmid is not in the bacteria yet. If you're trying to get it in there I suppose electroporation would work.
Nov 14 2012 10:43AM
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SkierPT
Posts: 2176 - Karma: 4,720
I guess we are supposed to make a large scale prep of the bacteria? does that sound right?
Nov 17 2012 9:55PM
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vandersteeze
Posts: 2650 - Karma: 1,511
This video is relevant

Nov 17 2012 11:27PM
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